Generation of K14 promoter-driven ATR-kd transgenic mice. (A) Schematic of ATR-kd transgene. FLAG-tagged, human ATR-kd was inserted into the polylinker (PL) region of the human K14 promoter/enhancer (P/E) construct containing a β-globin intron. Asterisk indicates the mutation site for inactivation of ATR kinase activity (Asp²⁴⁷⁵ → Ala²⁴⁷⁵). Solid arrows indicate the primer set used to detect the transgene in genomic DNA (B). Dashed arrows indicate the primer set used to quantify mRNA expression of the transgene (C). (B) ATR-kd transgene (ATR-kd Tg) is detected in genomic DNA from transgenic (Tg) mice, not from littermate controls (Ctrl), via PCR amplification and agarose gel analysis. (C) ATR-kd transgene mRNA is expressed in transgenic (Tg) mice, but not in transgene-negative littermate controls (Ctrl), as revealed by real-time RT-PCR analysis. (Left) Amplification plot of ATR-kd transgene. cDNA containing ATR-kd transgene sequence was used as positive control (cDNA). Samples from littermate controls (Ctrl) and negative control samples (no template) showed no amplification after 40 cycles. (Right) Amplification plot of the RNA quality control gene Gapdh. (D) FLAG-tagged ATR-kd protein is expressed in ATR-kd transgenic (Tg) mouse keratinocytes. Lysates (500 μg of total protein) of primary keratinocytes isolated from Tg mice and transgene-negative littermate controls (Ctrl) were used for immunoprecipitation (IP) with anti-FLAG antibody, followed by immunoblot (IB) analyses with the indicated antibodies. Input samples (15 μg of total protein) were from the same cell lysates but were not immunoprecipitated. The anti-ATR antibody used detects both human and mouse proteins.

ATR-kd transgene delays tumor onset and suppresses UV tumorigenesis. (A) UV irradiation timeline indicating cumulative UVB dosage. Mice were irradiated with 250–2,500 J/m² of UVB light per day, thrice weekly. (B) Percentage of tumor-free mice is shown for each group: ATR-kd transgenic (Tg) mice or transgene-negative littermate controls (Ctrl). All mice in both groups were Xpc^−/−. Kaplan–Meier curves were compared by using the logrank test for statistical significance. (C) Tumor onset is delayed in ATR-kd transgenic mice. Mean number of weeks of UV treatment required for developing an initial tumor is shown. (Error bars = SEM.) (D) ATR-kd transgene suppresses UV-induced tumor development. Mean number of tumors per mouse is shown up to 19 wk, the point when some mice with advanced tumors were killed and the cohort was no longer complete. (Error bars = SEM.) Statistical significance in mean number of tumors per mouse between the groups was as shown at the indicated time points: *P ≤ 0.05, **P < 0.01. (E) Correlation of visible tumor features with histological tumor invasiveness in a randomly selected subset of tumors at time of euthanasia. Before histologic examination, 47 tumors (22 from ATR-kd transgenic mice and 25 from transgene-negative littermate controls) were segregated into five groups as shown based on size, bleeding, and ulceration/cratering/erosion. Larger tumors (≥3 mm) with bleeding and/or ulceration/cratering/erosion were presumed to be invasive SCCs. Indeed, all 34 tumors that had visible features associated with invasive SCCs were verified pathologically to be invasive SCCs. The 14 tumors that did not meet clinical criteria for invasive SCC were found microscopically to be SCC in situ (n = 7), keratoacanthoma (n = 5), epidermal hyperplasia (n = 1), and invasive SCC (n = 1). (F) ATR-kd transgenic mice had fourfold fewer clinically defined invasive SCCs than transgene-negative littermate controls (Ctrl) did, although this result did not meet statistical significance (P = 0.071). Mean number of invasive SCCs per mouse after 23 wk of UV treatment is shown. (Error bars = SEM.)

Primary keratinocytes from ATR-kd transgenic mice demonstrate ATR signaling inhibition and augmentation of UV-induced apoptosis. (A) ATR-kd inhibits UV-induced phosphorylation of Chk1. Primary mouse keratinocytes isolated from ATR-kd transgenic (Tg) mice and transgene-negative littermate controls (Ctrl) were treated with medium or caffeine (3 mM) for 30 min before UV irradiation. Cells were irradiated with 75 mJ/cm² of UVB light and harvested 1 h after UV exposure for immunoblot analyses with the indicated antibodies. (B) ATR-kd does not inhibit doxorubicin-induced phosphorylation of ATM. Primary mouse keratinocytes isolated from ATR-kd transgenic (Tg) mice and transgene-negative littermate controls (Ctrl) were treated with medium or doxorubicin (1 μM) for 2 h and harvested for immunoblot analyses with the indicated antibodies. Arrowheads indicate same molecular size, corresponding to the ATM protein. (C and D) ATR-kd augments UV-induced apoptosis measured by sub-2N DNA content. Primary mouse keratinocytes were isolated from ATR-kd transgenic (Tg) mice and transgene-negative littermate controls (Ctrl). Cells were treated with medium or caffeine (3 mM) for 30 min before UV irradiation. Cells were irradiated with 75 mJ/cm² of UVB light, harvested 24 h later, and stained with propidium iodide for flow cytometry analysis. (C) Representative DNA content patterns of cells without caffeine treatment with percentage of sub-2N DNA content indicated. (D) Mean of percentage of sub-2N DNA content is shown (n = 4). (Error bars = SEM.)